Immunovectors for the intracellular and intranuclear transport

ABSTRACT

A product for coupling a biologically active principle with an immunovector, characterized in that the immunovector is capable of enabling the biologically active principle to be internalized into eukaryotic cells, and in that said immunovector has an affinity for the cell DNA to such an extent that it can transfer the biologically active principle into or to the immediate vicinity of the cell nuclei.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §3712 PCT/FR 96/01076filed Jul. 10, 1996 which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the active transfer of haptens,proteins, nucleic acids and other molecules into the nucleus ofeucaryotic cells. This invention is of major importance since it can beapplied to various fields, especially those of gene therapy andvaccines.

Gene therapy continues to be dependent on a considerable number ofparameters among which are the development of vectors capable oftransporting through the cytoplasm of these cells of the host organismactive principles endowed with predetermined specific properties intothe nuclei of cells of the organism in the absence of geneticalterations associated with the use of these vectors, and thenon-degradation of the biological activity of the active principlestransferred. It is known that so far all these conditions are far frombeing fulfilled (1).

Indeed, the current methods commonly used to transfer DNA into cells arethe following: general, non-selective methods which use the property ofDNA to coprecipitate with calcium phosphate or DEAE-dextran, oralternatively the direct introduction of DNA into cells under the effectof an electric field (electroporation). These methods are very toxic forthe cells, leading to a high mortality and a high variability accordingto the cells used. Other methods use targeting of the entry of the geneinto cells by receptors present on their membrane. The DNA may thenpenetrate into the cell via either a ligand specific for thesereceptors: asialorosomucoid (2), insulin (3) or transferrin (4), orantibodies specific for membrane constituents (5). The DNA/ligandcomplex penetrates into the cell by a process of endocytosis. Thetransfection is therefore limited by a substantial destruction of thecomplex in the lysosomal vesicles, and different methods have beenproposed to overcome these disadvantages, especially the blocking of thelysosomal compartment by chloroquine or the simultaneous addition ofadenoviruses which escape the endosomal compartment by destroying themembrane of the endocytosis vesicles (6).

The aim of the present invention is to provide a new type of vectorswhich are both more efficient and safer than the viral vectors whose usehas been envisaged until now.

The invention therefore relates to a product of coupling between abiologically active principle and one of these new vectors, hereinaftercalled “immunovectors”, the said product of coupling being characterizedboth by the capacity of the immunovector to allow the internalization ineucaryotic cells of biologically active principles linked covalently ornon-covalently to these immunovectors, and by their affinity for the DNAof these cells to such a point that the said immunovector is renderedcapable of transferring the biologically active principle immediatelyclose to the nuclei of these cells or into the nuclei of these cells.

These immunovectors preferably consist of antibodies or fragments ofantibodies capable of recognizing DNA sequences inside these cells, andto which biologically active principles may be covalently ornon-covalently linked, these antibodies or fragments of antibodiesbeing, in addition, capable of transporting in vitro and in vivo thesebiologically active principles through the membranes and the cytoplasmof these cells, and transferring them close to or even into the nucleusof these cells.

It is understood that in the present description the term “biologicallyactive principle” relates to any molecule, macromolecule or group ofmolecules having biological activity of the type in question.

The invention also relates to a method of transferring especiallyhaptens, proteins and/or nucleic acids into the nucleus of cells,particularly eucaryotic cells, this method being based on the use of theproperties of the said immunovectors.

The existence of antibodies capable of penetrating inside the nuclei ofhuman lymphocytes when these cells are incubated in vitro in a culturemedium containing a serum obtained from patients suffering fromdisseminated erythematous lupus (DLE) was reported for the first time byAlarcon-Segovia et al. in 1978 (7). Subsequently, the same teamdemonstrated that these antibodies are of the IgG isotype and arecapable of reacting with ribonucleic acids, free or complexed withproteins (8). Recently, this type of antibody was detected in MRLlpr/lpr lupus mice, but also in NZB mice having a haemolytic auto-immunedisease syndrome and even in normal BALB/c mice. Some monoclonalantibodies, prepared from the spleen of these mice, have proved capableof penetrating in vitro into the nucleus of cells maintained in culture(10-13). As in humans, it was noted that these monoclonal antibodieswere capable of recognizing nucleic acids. Furthermore, it was shownthat these antibodies are also capable, when they are injected intomice, of penetrating into several types of cells, ending up in theirnuclei (11).

The invention results from the discovery that this type of antibody orfragments of these antibodies could also be used as vectors, hereinafter“immunovectors” capable of transporting biologically active principles,such as haptens, proteins, and nucleic acids through the membranes andthe cytoplasm of the corresponding cells, and ensuring their transferinto the nucleus of the said cells.

These antibodies may be obtained in polyclonal form from a serum, inparticular from an animal previously immunized against nucleic acidfragments having the corresponding epitope, or in monoclonal form fromhybridomas secreting such antibodies.

Any type of bonding, chemical or otherwise, may be used to ensure thecoupling of an immunovector of antibody or antibody fragment type havingan affinity for the nucleic acids to the biologically active principle,for example a hapten or a nucleic acid, for the purpose of transportingit through the membranes and the cytoplasm of the cells, and to ensurethe transfer of these active principles into the nucleus.

Preferably, a chemical mode of coupling, allowing the formation ofcovalent or non-covalent bonds, will be used.

Preferred coupling products are those in which the immunovectors areselectable by a cellular penetration test comprising a first incubationof the immunovector of interest in the presence of cells in culture inthe nucleus of which the active principle capable of being associatedwith the immunovector has to be transported, followed, after fixing andpermeabilization of these cells, by another incubation with labelledanti-immunovector antibodies, and finally by a detection immediatelyclose to the nucleus or even inside the nucleus of the antigen-antibodytype immunologic reaction between the immunovector and theanti-immunovector antibody.

Among the preferred immunovectors of the present invention, there may bementioned the antibodies having an affinity for a nucleic acid, or afragment thereof, the latter retaining this affinity.

Antibodies also having the capacity to bind to the cells, in particularlymphoid cells, can also be used. The latter category of immunovectorsmay also be selected by a test, which may then also comprise theincubation of the immunovectors of interest with lymphoid cells, washingthe said lymphoid cells, incubating them with labelled anti-immunovectorantibodies, and determining the number of positive cells in eachpopulation.

In one embodiment, the lymphoid cells used are auto-immune mousesplenocytes exhibiting a lupus syndrome.

A preferred immunovector for the coupling product is chosen from amongmonoclonal IgG's, (Fab′)2 or (Fab′) fragments, or any polypeptidecorresponding to the site(s) of the antibodies involved in therecognition of the corresponding nucleic acid.

Preferably, this immunovector is an immunoglobulin, more particularly anIgG carrying an anti-DNA activity, and obtained from normal individuals.

In addition, this immunovector may be an IgG carrying an anti-DNAactivity and obtained from individuals presenting auto-immune syndromes,more particularly disseminated erythematous lupus syndromes.

In a specific embodiment of the invention, the immunovector coupled tothe active principle is a bi-specific antibody recognizing on the onehand the DNA, and on the other hand a protein such as Tat, Rev of theHIV retrovirus, as well as surface markers such as CD3, CD4, CD8, CD19and CD34.

Preferably, the biologically active principle coupled to theimmunovector is a molecule selected from especially nucleic acids,proteins especially enzymes, for example peroxidase, haptens especiallybiotin or fluorescein, enzyme activators or inhibitors and medicaments.

In a preferred embodiment, the coupled nucleic acid is a polynucleotide,the immunovector being an IgG, with the coupling being carried out viap-benzoquinone at the rate of one molecule of immunovector per 4molecules of polynucleotide.

A biologically active principle preferably used consists of a plasmidintended to integrate into the nucleus of the target cells for theexpression of a protein encoded by a gene contained in the said plasmid.When such a plasmid is coupled to an immunovector of the antibody type,having affinity for DNA, this immunovector is, preferably, previouslycoupled to an agent capable of inducing a compacting effect on the DNA,this agent being preferably polylysine.

Indeed, polylysine, which by virtue of its cationic properties iscapable of compacting DNA, promotes the transfection of cells. Thecoupling of polylysine to the immunovector, which is carried out withthe aid of a coupling agent, more particularly a carbodiimide such asEDC (1-(3-dimethylaminopropyl)-1′-ethylcarbodiimide), allows the DNA toreact therewith. This has the effect of inducing the liberation of theactive site of the antibody which may sometimes be masked when theantibody is coupled to an active principle of the size of a plasmid.

Other agents capable of having, by virtue of their cationic properties,a compacting effect on DNA (2-6) may also be coupled to the immunovectorto fulfil such a function.

More specifically, a biologically active principle preferably usedconsists of a gene intended to integrate in the genome of target cells,especially by homologous recombination, more particularly a “nude” DNAcontaining a nucleic acid sequence coding for a polypeptide originatingfrom bacterial or eucaryotic cells, fungal cells or viruses, thispolypeptide having vaccinating properties.

Advantageously, this active principle allows the immortalization ofselected types of cells, particularly macrophages, dendritic cells, Band T cells, and hematopoietic cells, especially of human origin.

Still more preferably, this biologically active principle is anantisense oligonucleotide allowing the inhibition of protein ornucleotide synthesis, for example in cells infectable by an HIVretrovirus, or in tumour cells.

Thus, the inventors have, on the one hand, tried to obtain, from thespleen of autoimmune mice (NZB×NZW)F1, having a lupus syndrome, IgGmonoclonal antibodies which have been selected for their capacity toreact with DNA but also for their capacity to penetrate as far as thenucleus of the cells. In parallel, polyclonal antibodies reacting withthe DNA and capable of penetrating into the nuclei of the cells havebeen isolated by affinity chromatography. This chromatography wasapplied either to a “pool” of sera of normal patients or to a normalindividual serum, preferably to sera obtained from patients sufferingfrom infections, particularly to a serum of normal patients sufferingfrom DLE, or to a mouse (NZB×NZW)F1 serum.

In a method of selection of immunovectors according to the presentinvention, the test of cellular penetration of the immunovectorscomprises a first incubation of eucaryotic cell lines in a mediumcomprising the said immunovectors preferably in increasingconcentration, then the fixing, and, if necessary, the permeabilization,or vice versa, of these cells, followed by an incubation of the saidcell lines with anti-immunovector antibodies preferably labelled withfluorescein or with peroxidase, and the localization of the antibodiesthus labelled close to the nuclei of the said cells or better stillinside the nuclei. The cell lines are especially chosen from amongfibroblasts, thymocytes or splenocytes.

Without the following reaction conditions having a limiting character,it may be mentioned that the first incubation is often carried out at37° C., for about 2 to 8 hours with immunovector concentrations of about1 to 70 μg/ml, on cell lines inoculated at a concentration ranging from5×10³ to 5×10⁶ cells per milliliter.

In one of the embodiments of the method, the cell line is a fibroblastline in exponential growth inoculated at a concentration of 2×10⁴ cellsper milliliter, or thymocytes or splenocytes of BALB/c mice, which aresuspended at the rate of about 10⁶ cells per milliter.

Moreover, the invention relates to a method of preparation ofimmunovector-molecule(s) coupling product, the immunovectors beingchosen from among antibodies, more particularly the IgG's obtainedaccording to the method of selection, (Fab′)2 or (Fab′) fragments, orany polypeptide corresponding to the site of the antibodies or fragmentsof antibodies involved in the transport of the molecules.

In the method of preparation of a coupling product according to theinvention, it is ensured that for each immunovector is coupled at leastone molecule of biologically active product, the said molecule beingpreferably covalently linked to the immunovector.

The following examples illustrate conditions in which haptens such asfluorescein and biotin, small molecules such as hormones, proteinspreferably enzymes, enzyme inhibitors or activators and medicines, forexample antivirals such as acyclovir or AZT, may be actively transportedthrough the cytoplasm of the treated cells and transferred into thenucleus of the said cells. In particular, fluorescein has been coupledto the free amino groups of the immunovectors via an activeisothiocyanate group and the biotin via an active succinimide ester. Thecoupling of the haptens and the like to the immunovector may be carriedout by means of other homo- or heterobifunctional bridging groups orreagents known in the literature, such as the imido esters andN-hydroxysuccinimidyl esters which are capable of reacting with theamino groups, for example derivatives of alkyl, haloaryl, haloacetyl andpyridyl disulphide groups reacting preferentially, maleimides, or bymeans of sulphydryl groups, carbodiimides, as well as molecules havingphotoactivable groups such as azidobenzoyl hydrazide (13).

In addition, the inventors have prepared products of coupling where theimmunovector is a bi-specific antibody against a target antigen,constructed either by the chemical route or by the genetic engineeringroute, and have in parallel immunized individuals, for example mice,with the said target antigens, and selected immunovectors which arebi-specific antibodies preferably reacting with the said targetantigens, such that the action of the immunovectors is directedspecifically.

Techniques relating to the synthesis of bi-specific antibodies haveespecially been described by Porstmann et al. in 1984 (16), a studyentitled “Development of a bispecific monoclonal antibody for use inmolecular hybridisation” having moreover been published in 1984 byAuriol et al. (17).

Advantageously, the bi-specific antibodies used in this methodrecognize, inter alia, the proteins Tat, Rev of the HIV retrovirus, aswell as surface markers CD3, CD4, CD8, CD19 and CD34.

Moreover, the present invention relates to a method for transferring anactive principle in the nuclei of selected eucaryotic cells,characterized by coupling this active principle with an immunovectorhaving both the capacity of allowing internalization of this activeprinciple in these eucaryotic cells and an affinity for the DNA of thesecells to such a point that the said immunovector is rendered capable oftransporting this biologically active principle immediately close to orinto the nuclei of these cells.

This biologically active principle may be covalently or non-covalentlycoupled to the immunovector.

Preferably, this method of transfer is characterized in that theimmunovector entering into the composition of this product is selectedfrom those which are selectable by a cellular penetration testcomprising a first incubation of the immunovector of interest in thepresence of cells in the nucleus of which the active principle capableof being associated with the immunovector has to be transported,followed, after fixing and permeabilization of these cells, by anotherincubation with labelled anti-immunovector antibodies, and finally thedetection immediately close to the nucleus or even in the nucleus of theantigen-antibody type immunologic reaction between the immunovector andthe anti-immunovector antibody.

In a preferred embodiment of the method of transfer according to theinvention, the immunovector used is formed of an antibody having anaffinity for a nucleic acid, or of a fragment of this antibody retainingthis affinity.

This immunovector used in this method is preferably selected from amongantibodies, preferably monoclonal IgG's, (Fab′)2 or (Fab′) fragments, orany polypeptide corresponding to the site(s) of the antibodies involvedin the recognition of the corresponding nucleic acid.

Advantageously, once the product of immunovector/active principlecoupling is prepared, it may be used for the intranuclear transfer ofother molecules. Thus, the fluorescein/immunovector conjugate tested maybe associated with an anti-fluorescein antibody coupled with a thirdmolecule, and thus transfer the said third molecule into the nuclei ofthe cells. Similarly, the biotin/immunovector conjugate may allow thebinding of an anti-biotin antibody or of avidin-streptavidin coupledwith a third molecule to be transferred into the nuclei.

In the present invention, an enzyme such as horseradish peroxidase wastransferred into the nucleus, but other proteins having variedbiological activities may also be used. Peroxidase coupled to theimmunovector via glutaraldehyde has also been used. However, othermethods known in the literature, such as those described in the case ofhaptens, may also be used.

As in the case of the hapten/immunovector conjugates, there may be used,in association with the protein/immunovector conjugates, an anti-proteinantibody coupled with a third molecule for the intranuclear transfer ofthe said molecule.

In the invention described herein, although a polynucleotide has beentransferred into the nucleus, a wide variety of nucleic acids havingappropriate biological activities may also be actively transferred atthe intranuclear level.

Thus, a method of transfer of active principles according to theinvention allows in particular the transfer of genes intended tointegrate into the genome of the target cells, especially by homologousrecombination, more particularly the transfer of “nude” DNA, it beingpossible for the latter especially to be used as “DNA vaccine”.

One of the homologous recombination techniques possible is thatdescribed by Mouellic et al. in 1990 (18).

Recent studies carried out by Whalen R. G. et al. have made it possibleto show the existence of an immune response following a DNA transfer.These studies, which have been the subject of patent application WO95/11307, were more particularly applied to the expression of monoclonalmolecules of the IL2 cytokine type (19).

In addition, this method of transfer makes it possible to introducenucleotide sequences involved in the immortalization of different celltypes, particularly macrophages, dendritic cells, B and T cells, andhaemotopoietic cells especially of human origin. As nucleotidesequences, there may be mentioned the oncogenic sequences or viralsequences associated with cell transformation phenomena.

It also allows the transfer of antisense oligonucleotides allowing theinhibition of protein or nucleotide synthesis, for example in cellsinfectible by a retrovirus such as HIV, or tumour cells.

In the present invention, the polynucleotide was coupled to theimmunovector via p-benzoquinone. However, other methods known in theliterature may also be used.

Moreover, the present invention also relates to the eucaryotic cellscontaining active molecules preferably at the nuclear level,characterized in that the said molecules cannot be naturallyincorporated into the nuclei of the said cells or have a weak expressionlevel in the said cells. These molecules are presented in these cellsimmediately close to their nuclei or into their nuclei, and are coupledto an immunovector characterized by its affinity for the DNA of thesecells, in the form of a coupling product according to the invention.

Among the cells to which the present invention relates, there areespecially the cells which can be infected by a virus or tumour cells.

Also entering within the framework of the present invention are thehybridomas producing the antibodies according to the present invention,as deposited at the CNCM on Jun. 30, 1995 under the numbers I-1605,I-1606 and I-1607.

In addition, the invention relates to a pharmaceutical composition,characterized in that it contains, in combination with a physiologicallyacceptable vehicle, a coupling product according to the invention inwhich the biologically active principle is a medicine or vaccinal activeprinciple and the immunovector is compatible with the host organism towhich the medicine is directed.

Also entering into the framework of the present invention is the use ofthe coupling product of the invention for the expression, in receivingcells, of a nucleotide sequence which is heterologous to the DNA of thehost.

EXAMPLES 1. Preparation of Immunovectors

A) Polyclonal Immunovectors

The human or murine IgG's are first isolated by passage of a mixture ofsera obtained from individuals suffering from disseminated erythematouslupus—or from lupus mice (NZB×NZW)F1—on protein A immobilized onSepharose (14).

The isolated IgG's are passed over a column of DNA immobilized oncellulose. The specific anti-DNA antibodies present in these IgG's areattached to this DNA-cellulose immunoadsorbent and eluted with a 20 μMsodium carbonate-bicarbonate buffer, pH 10, containing 5% dimethylsulphoxide (15). 1 to 2 mg of antibodies are thus isolated from 10 mg ofIgG. The eluted antibodies are dialysed, concentrated and stored at +4°C. until they are used.

B) Murine Monoclonal Immunovectors

Splenocytes obtained from lupus mice (NZB×NZW)F1 are fused with the X63myeloma according to the method of Kö hler and Milstein. The hybridomasproduced are tested by ELISA for the secretion of IgG and for theiranti-DNA activity. The anti-DNA IgG secreting hybridomas are subclonedat least twice and the clones which remain doubly positive(IgG+anti-DNA) are bulk cultured or alternatively ascites are preparedfrom these clones in mice. In a typical experiment, starting with thespleen of a mouse (NZB×NZW)F1, approximately 300 positive wellssecreting IgG's were obtained of which 60 were capable of reacting withDNA. After cloning, 20 clones secreted IgG recognizing DNA. Of these 20clones, approximately half secreted antibodies capable of penetratinginto the nucleus of the cells whereas the others were not capable ofthis (see C: test of intranuclear penetration of the immunovectors). Themonoclonal IgG's are isolated from culture supernatants or asciticfluids by precipitation with 45% ammonium sulphate followed, afterdialysis, by passage on protein A immobilized on Sepharose. Afterneutralization of the eluted IgG's, the preparations are dialysed,concentrated and stored at −20° C. until they are used.

The mouse antibodies mentioned above will be subsequently humanizedusing one of known techniques, for example that described by Riechmannet al. (20).

C) Selection of the Immunovectors

1) Test of Intranuclear Penetration of the Immunovectors

Two fibroblast lines—PtK2 obtained from kangaroo rat kidney and GMA-32obtained from hamster kidney—were mainly used. The slides carrying thefibroblasts in the exponential growth phase, inoculated at 2×10⁴cells/ml, 24 hours beforehand and cultured in RPMI 1640 or MEM medium(containing 10% foetal calf serum, 2 mM L-glutamine and 1% sodiumpyruvate) are incubated at 37° C. in renewed culture medium, containingselected quantities of immunovector (1 to 70 μg/ml) After 2 to 4 hoursof incubation, the cells are washed with PBS and fixed with eitherethanol for 10 minutes at −20° C., or with 0.2% glutaraldehyde and 2%formaldehyde in PBS for 20 minutes. After three washes with PBS, thecells are permeabilized for 20 minutes in PBS containing 0.2% bovineserum albumin and 0.5% saponin.

The cellular preparations are then washed with PBS and incubated for 45minutes at 24° C. with anti-mouse immunoglobulin (or anti-humanimmunoglobulin) rabbit or sheep antibodies labelled with fluorescein orwith peroxidase (20 μg/ml). After washing, the cellular preparationsincubated with the fluorescent antibody are examined under afluorescence microscope. The cellular preparations incubated with theperoxidase-labelled antibody are first incubated in the cytochemicalsubstrate of peroxidase (diaminobenzidine (DAB)+H₂O₂) and, afterwashing, the preparation is examined under an optical microscope (14).The number of positive cells is counted.

As described above, mouse thymocytes were also used to test thepenetration of the immunovectors into the nucleus. Suspensions ofthymocytes were prepared from BALB/c mouse thymus. The thymocytes, at aconcentration of 1×10⁶ cells/ml, are incubated at 37° C. for 3 hours ina culture medium containing increasing quantities of immunovector (1 to70 μ/ml). After washing and fixing, the lymphocytes are treated like thepreparations of fibroblasts above for the intranuclear detection of theantibodies.

2) Test of Attachment of the Anti-DNA Antibodies to the Lymphoid Cells

To demonstrate a reaction with the cell membranes, 10⁶ mouse thymocytesor splenocytes were incubated at cold temperature for 45 minutes with0.1 ml of different monoclonal antibodies diluted in a solution ofbovine albumin at 0.1% containing 0.2% sodium azide. After washing, thecells are incubated with fluorescent anti-mouse IgG antibodies for 45minutes at cold temperature. After washing, the cells are examined byFACS and the number of positive cells determined in each population.

Of the 20 monoclonal antibodies examined, approximately half secreteantibodies which are capable of penetrating into the nucleus of thecells whereas the other half do not have this capacity. A correlationwas able to be established between the monoclonal antibodies penetratingwith a high efficiency into the nucleus of the cells (number of labelledcells, limiting dilution to obtain a labelling) and their ability tolabel the thymocytes and the splenocytes.

II Preparation of Immunovectors Carrying Haptens, Proteins or NucleicAcids

A) Preparation of F(ab′)2 and Fab′ Fragments of Immunovectors

The F(ab′)2 fragments of the immunovectors are prepared according todescribed methods involving proteolysis with pepsin followed byreduction by cysteine to obtain the Fab′ fragment (14). Thus in 5 ml of0.1 M citrate-citric acid buffer, pH 3.5, containing 5 mg ofimmunovector, 150 μg of pepsin are added and the mixture is incubatedfor 2 hours at 37° C. The medium is adjusted to pH 8 and the preparationfiltered on a protein A-Sepharose column in order to remove theundigested IgG's. After dialysis against PBS, this F(ab′)2 preparationis stored at −20° C. until it is used. To obtain the Fab′ fragments,cysteine is added to a final concentration of 0.02 M to the F(ab′)2preparation. After 10 minutes of incubation at 37° C., 0.04 Miodoacetamide is added and the mixture is incubated for 30 minutes. ThisFab′ preparation is dialysed against PBS and stored at −20° C. until itis used.

B) Immunovectors/haptens

Coupling to Biotin

2 μl of a 0.1 M solution of d-biotin-N-hydroxysuccinimide ester indimethylformamide (1 mg of the active ester in 30 μl ofdimethylformamide) are added to 0.5 ml of 0.1 M phosphate buffer, pH 7,containing 1 mg of antibody. The solution is left for 1 hour atlaboratory temperature and dialysed against PBS at +4° C. overnight.

Coupling to Fluorescein

20 μl of a solution of fluorescein isothiocyanate in dimethyl sulphoxide(10 mg/ml) are added to 1 ml of a 0.1 M solution of sodium carbonatecontaining 1 mg of antibody. The solution is left for 3 hours atlaboratory temperature and dialysed against PBS at +4° C.

C) Immunovectors/proteins

Coupling with Peroxidase

Ten milligrammes of peroxidase are dissolved in 0.2 ml of 1%glutaraldehyde in 0.1 M phosphate buffer pH 6.8. After incubation atlaboratory temperature for 18 hours, the solution is filtered on aSephadex G25 column (0.9×60 cm) equilibrated with 0.15 M NaCl to removethe excess glutaraldehyde. To this activated peroxidase solution, thereis added 1 ml of a 0.15 M NaCl solution containing 5 mg of antibody and0.2 ml of 1 M carbonate-bicarbonate buffer pH 9.5. The solution isstored at +4° C. for 24 hours and then supplemented with lysine to thefinal concentration of 0.1 M, and 25 then dialysed against PBS at 4° C.

D) Immunovectors/nucleic Acids

Polynucleotides

The polynucleotide used was composed of 15 nucleotides and carried afluorescein in 5′ and a free NH₂ group in 3′. It was prepared accordingto conventional methods of nucleic synthesis. This nucleotide wascoupled to the immunovector via p-benzoquinone (14). 0.1 ml of ethanolcontaining 3 mg of p-benzoquinone is added to 0.4 ml of 0.1 M phosphatebuffer pH 6 containing 1 mg of immunovector (whole molecule, F(ab′)2 orFab′). After an incubation of one hour at laboratory temperature, thepreparation is filtered on a Sephadex G-25 column. The fractioncontaining the activated immunovector is supplemented with thepolynucleotide in a ratio of one molecule of immunovector to 4 moleculesof polynucleotide, and the solution is adjusted to pH 9.2 withcarbonate-bicarbonate buffer. After 18 hours incubation at laboratorytemperature, the reaction is stopped by addition of lysine to the finalconcentration of 0.1 M, followed by dialysis against PBS. Thispreparation is stored at +4° C. until it is used.

Plasmids

Two plasmids were tested, a first carrying the vimentin promoterupstream of the gene encoding the SV40 T, t antigens (pHuVim 830 T,t)(21) and a second carrying the luciferase gene (22) under the control ofa cytomegalovirus promoter (pCMV-Luc) (5).

These plasmids are maintained in the E. coli strain and are preparedafter a bacterial culture by the standard lysis method in the presenceof detergent and in alkaline medium. The plasmids are then purified bychromatography on a resin column (Qiagen Plasmid Kits).

The immunovectors J-20.8 and F-14.6 are used for this work. Theseantibodies are prepared by the method of preparation of monoclonalimmunovectors previously described in paragraph I.B/. The antibodies arecoupled to poly-L-lysine with the aid of a coupling agent, especially acarbodiimide, such as EDC(1-(3-dimethylaminopropyl)-1′-ethylcarbodiimide). In some cases,polyclonal IgG's are added to the anti-DNA antibody in a 10:1 ratio soas to increase the concentration of IgG in the medium and thereby topromote the coupling with the polylysine.

2 mg of monoclonal antibody (J-20.8 or F-14.6) in 1 ml of PBS or ofconcentrated polyclonal IgG's at 20 mg/ml are dialysed overnight againsta 10 mM MES buffer, pH 5. Two mg of poly-L-lysine (MW=18,000) aredissolved in 1 ml of this same buffer and then supplemented with 0.2 mgof EDC (in 50 μl of MES buffer) for 30 seconds. The poly-L-lysinesolution is then added to the antibody/EDC mixture and the incubation iscontinued for 2 hours.

the preparation is then filtered on a protein A-Sepharose column inorder to separate the excess poly-L-lysine from the antibodiesconjugated to the polylysine which are eluted at pH 3 under the usualconditions, neutralized and dialysed against PBS.

III. Examples of Transfer of Substances into the Nuclei of Cells byImmunovectors Associated with These Substances

A) Transfer in vitro of Fluorescein

Fibroblasts of the GM A-32 line in culture on glass coverslips areincubated at 37° C. for 2 to 4 hours in RPMI culture medium containingincreasing quantities of monoclonal immunovectors (J-20.8 antibody orFab′2 fragments) labelled with fluorescein. At the end of this time, thecells are washed and fixed as described in IC1. After inclusion inMowiol medium, they are examined under a fluorescence microscope.Practically all the nuclei of the fibroblasts show a fluorescentlabelling. On the other hand, the nuclei of fibroblasts incubated with acontrol monoclonal antibody Ig 2a without anti-DNA activity, which doesnot penetrate to the nuclei, do not exhibit any fluorescence (FIGS. 1and 2).

B) Transfer in vivo of Fluorescein into Mouse Peripheral Lymphocytes

One mg of immunovector (monoclonal antibody C-2.1 or F-4.1 or controlantibody (monoclonal antibody G-14) labelled with fluorescein isinjected into two mice in an amount of 0.2 ml intraveneously and 0.3 mlintraperitoneally. After 5 hours, the mice are bled and sacrificed andthe circulating blood lymphocytes are analysed by FACS. It is noted that60% of the peripheral blood lymphocytes obtained from the mouse injectedwith the immunovector are fluorescent whereas none of the lymphocytesfrom the control animal are (FIG. 3). Microscopic examination shows afluorescence at the level of the nuclei in the majority of the cells.

C) Transfer of Biotin

Fibroblasts of the PtK2 line (10⁵/ml) cultured for 24 hours beforehandare incubated in a complete RPMI culture medium with human anti-DNApolyclonal IgG's labelled with biotin in increasing quantities (5-100μg). After 3 hours, the cells are washed, fixed and permeabilized asdescribed in IC. The cells are then incubated with RPMI containing 1μg/ml of peroxidase-labelled streptavidin. After one hour, the cells arewashed three times with PBS and the peroxidase associated with the cellsis revealed using the DAB+H₂O₂ medium. The preparations are included inMowiol and examined under an optical microscope. A large number ofnuclei of the fibroblasts incubated with anti-DNA IgG are positivewhereas the cells incubated with IgG's obtained from normal individualsand labelled with biotin are negative.

D) Transfer of Peroxidase

Under the conditions defined in paragraph IIIC, the PtK2 fibroblasts areincubated with increasing quantities of Fab′ fragments of animmunovector (antibody J-20.8) labelled with peroxidase. After 3 hours,the cells are washed three times with PBS and fixed for 20 minutes with0.2% glutaraldehyde and 2% formaldehyde in PBS. After washing, theperoxidase activity is revealed by the coloured DAB+H₂O₂ test and thepreparations are examined under an optical microscope. A largeproportion of nuclei of the fibroblasts incubated with the Fab′fragments of the J-20.8 antibody are positive for peroxidase, whereasthose incubated with the control antibody 48.9 are negative.

E) Transfer of Polynucleotide Labelled with Fluorescein

3×10⁶ splenocytes, prepared from BALB/c mouse spleen, are incubated in 1ml of RPMI containing 40 μg/ml of immunovector (J-20.8) or of its Fab′fragment covalently coupled to the polynucleotide. After three hours ofincubation at 37° C., the cells are washed with PBS, fixed in 4%paraformaldehyde and examined under a microscope. Eight to 10% of thecells show an intranuclear fluorescence (FIG. 4).

F) Transfer of Plasmid

The transfection efficiency was evaluated by demonstrating the synthesisof the proteins encoded by these genes, either with the aid of anti-Tantigen antibodies coupled to peroxidase, or by a luminometric assay ofthe activity of luciferase on its substrate, luciferin.

The cells used are fibroblasts of the GMA-32 line and Hep 2 carcinomacells. They are cultured in a complete medium (RPMI 1640 mediumcontaining 10% foetal calf serum, 2 mM L-glutamine, 1% sodium pyruvateand antibiotics), at 37° C. of 5% CO₂.

Plasmid pHuVim 830 T,t: The Hep2 cells are inoculated the day before inan amount of 2×10⁴ cells in 0.5 ml of complete medium per well of a24-well plate. For the transfection, the medium is removed and replacedwith 0.3 ml of complete medium containing 20 μg ofantibody-poly-L-lysine and 2 μg of plasmid, or 20 μg of nativeantibodies and 2 μg of plasmid or 2 μg of plasmid alone. After 6 hours,the medium is changed and the culture is continued by changing themedium every two days and by subdividing the cells into two ifnecessary. The transfection efficiency is tested at variable times.

Plasmid pCMV-Luc: The GMA-32 cells are inoculated the day before in anamount of 7 to 10×10⁴ cells/0.5 ml of complete medium per well of a24-well plate of complete culture medium. For the transfection, themedium is removed and replaced with 0.5 ml of complete medium containing8 μg of J-20.8/polylysine or of F-14.6/polylysine, or 20 μg of J-20.8polyclonal IgG and 2 μg of plasmid or 2 μg of plasmid alone. After 6hours, the medium is changed. The transfection efficiency is tested 24hours after the start of transfection.

Control of Transfection

Plasmid pHuVim 830 T, t: The synthesis of the T antigen in the nucleusof the transfected cells is demonstrated by an immunocytochemicalmethod. The cells are washed 3 times with PBS and then fixed for 10minutes in methanol at −20° C. They are then incubated with the anti-Tantigen antibody coupled to peroxidase for 1 hour. After washing, theperoxidase is revealed with the DAB+H₂O₂ mixture. In the well incubatedwith the J-20.8 polylysine and plasmid complex, isolated cells and a fewclusters of cells have a nucleus which is intensely coloured brown after48 hours and after 2 weeks. The control with the native antibody or theplasmid alone is negative.

Plasmid pCMV-Luc: The transfection efficiency is demonstrated by theluciferase synthesis detectable in the lysates of the transfected cells.This enzyme catalyses the oxidation of luciferin which results in aproduct detectable in a luminometre. After the culture, the cells arewashed in PBS and then lysed in 25 mM tris-phosphate buffer, pH 7.8,containing 8 mM MgCl₂, 1 mM DTT, 1% triton X100, 1% BSA and 15%glycerol. The lysate is assayed in a luminometre by automated additionof a solution of luciferin (0.25 mM) and of ATP (1 mM). An aliquot ofthe same lysate is assayed for its protein concentration using aCoomassie (Bio-Rad Protein Assay) reagent. The results are expressed inunits (RLU) per mg of proteins. As shown in the table, a gene transfertakes place in the presence of the antibody preparationsJ-20.8/polylysine and F-14.6/polylysine whereas the IgG/polylysinepreparations have no effect.

The transfection efficiency for the same antibody/plasmid ratio is 10times higher with the J-20.8 preparation than with F-14.6. Furthermore,the addition of concentrated polyclonal IgG's during the coupling topolylysine appears to increase the transfection efficiency since 2 μg ofJ-20.8 (complex 20:0.5) of the J-20.8-IgG/polylysine preparation giveresults of the same order of magnitude as 8 μg (complex 8:0.5) of theJ-20.8/polylysine preparation.

The entire results obtained are summarized in the following table:

Antibody/plasmid Assay Immunovector ratio μg/μg RLU/mg × 10⁴ J-20.8-IgG/20:0.5 5.8 polylysine 20:0.5 63.00 J-20.8/ 8:2  2.00 polylysine 8:1 15.00 8:1  38.00 8:0.5 13.00 F-14.6/polylysine 8:0.5 1.3 IgG/polylysine20:0.5  <0.1 20:0.5  <0.1 Plasmid 2 <0.1 alone (μg) 1 <0.1 0.5 <0.1

In the preceding text, the preferred immunovectors consisted essentiallyof the anti-DNA antibodies or of fragments of these antibodies, with theproviso that these fragments retain the site of recognition for thewhole DNA. Naturally, it goes without saying that the immunovectorswhich can be used within the framework of the invention may be preparedin any other way, as long as they would also allow the transport of thebiologically active principle which would be associated therewiththrough the membranes of these cells and their cytoplasm and itstransfer close to the nucleus of the cells, or even inside this nucleus.

By way of examples of such immunovectors, there may be mentionedconjugates between a nuclear protein, for example a histone, a proteinhnRNP, a polymerase or a factor associated with this polymerase, and theactive product, this nuclear protein being itself (unless it is capable,on its own, of bringing about the internalization and the transfer of abiologically active principle into the nucleus of the cells, conjugatedto the anti-cell membrane receptor antibody or to any other moleculeallowing the internalization into the cell of the conjugate thusproduced. As long as this conjugate is capable of diffusing as far asthe nucleus of the cells and that, moreover, it may in turn transportand transfer, as was defined above, a biologically active principlewhich would be coupled to this conjugate, it constitutes an immunovectorentering within the framework of the present invention.

The selection techniques which have been described above for the choiceof efficient immunovectors for the transfer of an active principle tothe nucleus of the cells are equally applicable to the selection of theabovementioned conjugates.

Persons skilled in the art will understand that an additional criterionfor choice may, at least for some of the conjugates used, lie in theabsence of an undesirable action of the nuclear protein which itcontains with the cell function. It is in fact to be noted that only thepart of the intranuclear protein carrying its site of recognition of thecorresponding DNA is essential for the embodiment in accordance with theinvention.

LEGEND TO THE FIGURES

FIG. 1: Transfer of fluorescein “in vitro”

Labelling of the nuclei of the GMA 32 fibroblasts with an immunovectorlabelled with fluorescein (antibody J-20.8) in A and B (×100magnification).

C: Absence of labelling with the fluorescent control antibody (×100).

D: Another field observed at low magnification (×40).

FIG. 2: Same preparation as that of FIG. 1A analysed under a confocalmicroscope. The GMA 32 fibroblasts are labelled essentially in thenucleus. A total fluorescence may be noted in FIG. 2A. FIG. 2Bcorresponds to the analysis of the fluorescence intensity.

FIG. 3: Transfer of fluorescein “in vivo” FACS analysis of mouseperipheral blood cells injected 5 hours beforehand in FIG. 3A, thefluorescent control antibody and in FIG. 3B with a fluorescentimmunovector the antibody (J-20.8). Histogrammes representing on thex-axis the fluoresence intensity (in arbitrary units) and on the y-axisthe number of cells.

FIG. 4: Analysis of the peripheral blood cells (of FIG. 3) by confocalmicroscopy. A total fluorescence can be noted in FIG. 4A. FIG. 4Bcorresponds to analysis of the fluorescence intensity.

FIG. 5: Transfer of a nucleotide “in vitro” Confocal microscopyexamination of a mouse splenocyte into which has penetrated the Fab′fragment of an immunovector (antibody J-20.8) coupled to a fluorescentnucleotide. A total fluorescence is observed in FIG. 5A. FIG. 5Bcorresponds to analysis of the fluorescence intensity.

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What is claimed is:
 1. A product comprising an antibody which recognizesan epitope contained in a nucleic acid, said antibody is coupled to abiologically active principle, wherein said biologically activeprinciple is a protein, or a nucleic acid; and wherein said antibody isproduced by a hybridoma selected from the group consisting of I-1605,I-1606, and I-1607.
 2. The product of claim 1, wherein said nucleic acidis an antisense oligonucleotide.
 3. The product of claim 1, wherein saidnucleic acid is a recombinant plasmid.
 4. The product of claim 1,wherein said protein is an enzyme, an enzyme inhibitor, or an enzymeactivator.
 5. The product of claim 1, further comprising polylysine. 6.A method of preparing the product of claim 1, comprising coupling theantibody with said biologically active principle.
 7. The method of claim6, wherein said coupling is non-covalent.
 8. The method of claim 6,wherein said coupling is covalent.
 9. The method of claim 6, whereinsaid coupling is mediated by a chemical coupling agent.
 10. The methodof claim 9, wherein said chemical coupling agent is selected from thegroup consisting of gluteraldehyde, imido esters, N-hydroxysuccinimidylesters, maleimides, azidobenzoyl hydrazide, carbodiimide, andbenzoquinone.
 11. The method of claim 10, wherein said carbodiimide isEDC (1-(3-dimethlyaminopropyl)-1′-ethylcarbodiimide).
 12. A method oftransferring a biologically active principle of interest into a cellcomprising: coupling an antibody which recognizes an epitope containedin a nucleic acid with the biologically active principle thereby forminga coupled biologically active principle; and incubating the coupledbiologically active principle with the cell, wherein said coupledprinciple is transferred through the cell membrane and into the cell andwherein said biologically active principle is a protein, or a nucleicacid.
 13. The method of claim 12, wherein said nucleic acid is anantisense oligonucleotide.
 14. The method of claim 12, wherein saidnucleic acid is a recombinant plasmid.
 15. The method of claim 12,wherein said protein is an enzyme, an enzyme inhibitor, or an enzymeactivator.
 16. The method of claim 12, wherein said coupled biologicallyactive principle is further coupled to polylysine.
 17. The method ofclaim 12, wherein said antibody is produced by a hybridoma selected fromthe group consisting of I-1605, I-1606, and I-1607.